Method for determining the distinctive nutritional requirements of a patient

ABSTRACT

The present invention relates to a method for determining the distinctive nutritional requirements of a patient with specific nutritional needs and providing a composition meeting the distinctive nutritional requirements of said patient.

FIELD OF INVENTION

The present invention relates to a method for determining thedistinctive nutritional requirements of a patient with specificnutritional needs and providing a composition meeting the distinctivenutritional requirements of said patient.

BACKGROUND

A number of parameters or compounds define the nutritional status of asubject. For example, nutrients, micronutrients, and other compounds arefound in certain concentrations in a fluid or tissue of the subject. Anumber of diseases change the concentrations of these compounds orvalues of these parameters, due to increased utilization of thesecompounds to fight against the disease, metabolic changes, and/orsuboptimal dietary management of the patient. As a result the subjectsuffering from such a disease is malnourished because the relevantparameters and compounds are no longer in the range found in a healthysubject and lead to nutritional deficiencies, like improper provision ofstructural components, insufficient energy supply, or a lack offunctional components. Thus, the subject suffering from a diseaseaffecting the nutritional status can benefit from a nutritionalintervention addressing the distinctive nutritional requirements of saidsubject. Providing the subject with a nutritional composition comprisingnutrients and micronutrients in amounts that reestablish the metabolic,physiologic and functional equivalence of the nutritional status of ahealthy subject would therefore be required.

A particular example of such a disease where the subject exhibitsdistinctive nutritional requirements is inflammatory bowel disease(IBD).

The role of nutrition in IBD gathers high interest, especially inpediatric Crohn's Disease (CD), where studies have shown that exclusiveenteral nutrition (EEN) can induce remission in mild to moderate diseasecomparable to corticosteroids. Thus, nutritional interventions offeredin addition to the standard of care (SoC) are an appealing option for asafe long-term disease management. Malnutrition is common in pediatricand adult patients with IBD, especially in those with CD, and typicallymanifests as protein-energy deficit yielding to general weight loss,and/or vitamin/mineral deficiencies. In general, poor dietary intakesecondary to postprandial abdominal pain and diarrhea is the most commoncause of malnutrition in IBD. The degrees of malnutrition depend on theduration, severity and extend of the disease, as well as loss offunction due to bowel resection or fibrosis. IBD patients have also beenreported to have fat and muscle mass depletion; and micronutrientdeficiencies also occur with mild disease or in remission phase.

Beyond malnutrition associated nutrient deficiencies, nutrition is alsoconsidered as an effective approach to the maintenance of the remissionphase and particularly to maintain the mucosal health. Intestinal mucinsforming the mucus gel and protecting the intestinal epithelium have beensuggested of crucial importance to restore epithelial health aftermucosal injury in IBD. The body capacity to maintain adequate mucinsynthesis is directly related to the bioavailability of some specificamino acids. Intestinal inflammation is known to increasegastrointestinal threonine uptake and mucin synthesis in enterally fedminipigs. Therefore, under inflammatory conditions as in IBD, specificamino acids could become conditionally essential to sustain mucinsynthesis justifying thus for their nutritional specific enrichment.

Accordingly, there is the need for a method identifying the distinctivedietary needs of patients suffering from a disease(s) or clinicalcondition, with a nutritional status that is different from thenutritional status of a healthy subject.

SUMMARY

It is an object of the invention to provide a new method for determiningthe distinctive dietary needs of a patient with a nutritional statusthat is different from the nutritional status of a healthy subject.

The invention relates to an in vitro method for determining thedistinctive disease related nutritional requirements of a subjectsuffering from a disease by: a) first determining in a sample of asubject suffering from the disease a profile of the statuses of markers(including nutrients, micronutrients and/or their metabolites, and/or orbiomarkers, or any combination thereof) otherwise referred to as a“nutritional profile”; b) second determining in a sample from a healthysubject or from a patient with a different severity or stage of thedisease, a similar nutritional profile of the statuses of the samemarkers (e.g. the same nutrients, micronutrients and/or theirmetabolites and/or biomarkers) determined in step a)., and c) thirdcomparing the nutritional profiles determined in step a). and b)., andthereby determining the distinctive nutritional requirements for apatient suffering from the disease

The method makes it possible to determine the nutritional profile, anddistinct nutritional requirements, of patients suffering from aparticular disease, and of patients with different levels of severity,or at different stages, of a same disease. Based on these identifiednutritional requirements a nutritional composition can be manufacturedcomprising nutrients and micronutrients in an amount capable ofrestoring or improving the nutritional profile of the patient sufferingfrom the disease towards the nutritional profile of a healthy subject.

Nutritional compositions are provided comprising nutrients andmicronutrients in an amount capable of reestablishing, in the patientsuffering from the disease, the nutritional profile of a healthysubject, or a of patient with an improved condition (i.e. lower diseaseseverity).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Diagram displaying the methodological approach towards definingdistinctive nutritional requirements. Nutritional profile or nutritionalstatus is used to quantify distinctive nutritional/nutrient requirementsassociated with a particular disease. Nutritional profile is indicativeof overall nutritional status and is measured through a series ofnutrient and micronutrient values and/or their related status markersthat are measured across patients and healthy populations. Nutritionalprofile can be correlated with clinical information (including clinicalmarkers) relative to the state of the disease (relapse, remission,degree of severity). Nutritional profiles are compared between patientand healthy groups, and/or within patient groups defined relatively todisease activity, severity or stage. If differences in nutritionalprofiles are observed between patients and healthy controls, thendistinctive nutritional/nutrient requirement (DNR) is defined. In FIG.1, “NP” is nutritional profile; “Da” is high disease activity orseverity or latest disease stage; “Db” is low disease activity orseverity or early disease stage; “Dc” is disease free or healthycondition; “#” is difference in Nutritional Profile; and “NP(Da)”,“NP(Db)”, “NPDc” are the Nutritional profiles of Da, Db and Dcrespectively.

FIG. 2. Diagram illustrating figuratively use of DNR to nutritionalcomposition of a product that aims at recovering nutrient levels asappropriate to alleviate symptoms, sustain remission and improve qualityof life of patients with a clinically proven efficacy. In FIG. 1, “NP”is nutritional profile; “Da” is high disease activity or severity orlatest disease stage; “Db” is low disease activity or severity or earlydisease stage; “Dc” is disease free or healthy condition; “#” isdifference in Nutritional Profile; and “NP(Da)”, “NP(Db)”, “NPDc” arethe Nutritional profiles of Da, Db and Dc respectively.

FIG. 3. Threonine and isoleucine metabolic products

FIG. 4. Determining threonine specific requirements in IBD throughcomparative analysis of threonine and other amino acid oxidation index

DEFINITIONS

“Nutritional status” relates to the quantifiable body status of a personor a population group (cohort). The nutritional status relates to theirstate of nourishment (the consumption and utilization of nutrients). Inthe present invention, the nutritional status is quantified usingmarkers indicative of said nutritional status, in particular,biological, biochemical, physiological markers, or other markersdetermined in a sample of a subject.

“A nutritional profile” relates to a set of quantitative measurements ofnutrients and micronutrients or of their relative status markers thatare to be determined in a sample (biological fluids such as blood redblood cells, blood plasma, blood serum, urine, tissue . . . ), and thusrequires that measurements of several, at least, two nutrients,micronutrients or relative status markers is determined.

A “marker” is a quantifiable parameter that represents oneparameter/marker in a profile or set of markers. The quantification ofsaid marker is the status of said marker. This parameter can directlyrelate to the amount of a certain compound, like a nutrient (e.g.protein, amino acid etc.) or a micronutrient (vitamin, element includingmineral etc.). The marker can, however, also relate to a value that isderived from the mathematical combination of nutrient and micronutrientconcentrations and/or status markers in the sample. The marker can alsobe functional markers, and inter alia relate to certain physiologicalactivities (e.g. enzyme activity) or physiological status (oxidativestress status).

“Distinctive nutritional requirements” (DNR) are those nutritionalrequirements that differ in a diseased subject and a healthy subject.For example, in the sample of a diseased subject the nutritional profileor the quantitative analysis of a profile of nutrients might bedifferent compared to the nutritional profile of a healthy subject.Under such circumstances, the observed differences in the nutritionalprofile can be used to identify and quantify disease-specific nutrientrequirements.

“Amino acids in free form” are amino acids that are comprised in acomposition as free amino acids not bound to other compounds, like otheramino acids, and are thus not contained in peptides or proteins.

Hence, “amino acids in bound form” are amino acids being part ofpeptides, proteins, or bound to other compounds.

“Protein amino acids” are those amino acids that are found in naturallyproduced proteins, including those that are used by the translationmachinery to produce proteins and those that that are modified inproteins subsequently to translation.

“Non-protein amino acids” are those amino acids that are not found innaturally produced proteins but which are metabolites or structuralcomponents in cells and organisms.

DETAILED DESCRIPTION OF THE INVENTION

The section headings serve to clarify the subject matter and should notbe interpreted to limit the subject matter.

The concept of the invention is illustrated by FIG. 1.

The nutritional profile of a subject suffering from a disease (diseasedsubject, patient), e.g. in a cohort of patients, is determined andcompared to the nutritional status of a cohort of healthy subjects(healthy controls). The nutritional profile encompasses thedetermination of a profile (with at least two measurements) ofnutrients, micronutrients, and/or nutrient/micronutrient status markers.The status of particular markers (quantification of said markers) isdetermined in a sample of the healthy subject and the patient sufferingfrom the disease. The comparison of the nutritional profiles of patientswith the nutritional profiles of healthy subjects serves to identifydifferences in the levels of nutrients and/or their relative statusmarkers between diseased subjects and healthy controls that are used toidentify and quantify distinctive nutritional requirement in thediseased population. In addition, the comparison of nutritional profileswithin a patient population stratified relatively to the degree ofseverity or stage of a disease enables to identify set of nutrients andmicronutrients that are associated with lower disease activity orseverity or stage, associating this with improved clinical condition ofthe patients.

In the next step, it is possible to formulate nutritional compositions(products) that take into account the respective distinctive nutritionalrequirements. In addition, variations of the nutritional profile withina diseased patient population stratified relatively to the degree ofseverity or stage of the disease is also used to determine nutritionalcompositions (products) that aim to restore nutritional profiles ofpatients with higher disease activity or severity towards values ofnutritional profiles of patients with lower disease activity orseverity.

For example, in a patient where a reduced blood level of the amino acidthreonine has been observed, and/or alterations in the level of one ormore of its oxidation metabolite(s) (FIG. 3), relative to values ofthreonine, and/or its oxidation metabolite(s), in healthy subjects, thenutritional composition will either contain proteins, peptides in orderto adjust the dietary supply of threonine, or the free amino acidthreonine itself (FIG. 2). Said composition is to be administered to thepatient with the aim to enable the patients to match their specificrequirement for threonine. In this way, an increased demand of thediseased subject for threonine is met and the eventual deficiency withrespect to threonine is corrected.

Some diseases display different degrees of severity with associateddifferent nutritional needs. In such a case, the cohort of patientswhere the nutritional status is to be determined will be a cohort ofpatients displaying the same or similar severity grade of the disease.In this way, it is possible to identify the distinctive nutritionalrequirements for patients with a particular severity grade of thedisease and thus provide products adapted to that severity degree. Themethod will now be described in more details.

Method for determining the distinctive nutritional requirements of adiseased subject:

The invention relates an in vitro method for determining the distinctivedisease related nutritional requirements of a subject suffering from adisease characterized by distinctive nutritional requirements in thediseased subject over a healthy subject comprising the steps:

a. Determining in a sample of a patient suffering from the disease aprofile of statuses of markers (including nutrients, micronutrientsand/or their metabolites and/or biomarkers), indicating the nutritionalprofile of said patient,b. Determining in a sample of a healthy subject a profile of thestatuses of the same markers determined in step a, indicating thenutritional profile of the healthy subject,c. Comparing the profiles determined in step a. and b., and therebydetermining the distinctive nutritional requirements for nutrients in apatient suffering from the disease.

Various nutrients, micronutrients, and their relative metabolites ofstatus markers can be determined (quantified). For each marker itsstatus is determined, i.e. the presence of said marker or its value.Metabolic inter-connected nutrients, micronutrients or related markerscan be grouped for an integrated analysis and interpretation.

Depending on the determined nutritional profiles certain nutrient andmicronutrient deficiencies can be identified in the diseased patient.The identification of these deficiencies then allows to provide a set ofadequate nutrients or micronutrients in the nutritional composition withlevels properly adjusted to compensating those deficiencies.Administering this set of nutrients to the diseased subject can have theeffect that the nutritional profile in the diseased subject is recoveredtowards the nutritional profile a healthy subject.

The markers indicating the nutritional status of nutrients can be director indirect markers. Direct markers indicate, for example, the amount ofnutrients or micronutrients. Indirect markers can be derived fromdirectly determined markers, e.g. by their combination, or can bemetabolites or catabolites of the nutrients or micronutrients, and/orbiomarkers or catabolites of the nutrients or micronutrients; or canrelate to a particular physiological state in the body.

The indirect markers can relate to the determination of the relativestatus of the nutrients and micronutrients, i.e. the ratio of particularnutrients to other nutrients, the ratio of particular nutrients tomicronutrients, the ratio of particular micronutrients to particularmicronutrients, or a combination of the above.

In addition, indirect markers can relate to the determination offunctional markers in the diseased subject. Functional markers aremarkers relating to the status of physiological or biochemicalparameters indicating the health status of a subject. For example,functional markers can be measurements such as erythrocyte transketolaseactivity, erythrocyte glutathione reductase activity, oxidative stressstatus, or nitric oxide synthase activity. These functional markersdisplay a certain (reference) status in healthy subjects. In patientssuffering from a disease, these markers may different values thusindicating inappropriate nutrient or micronutrient status relatively tohealthy subjects. Appropriate provision of the diseased subject withappropriate set and levels of nutrients and micronutrients in anutritional composition can be used to restore levels of thesefunctional markers towards the levels measured in a healthy subject.

Samples:

The determination of the nutritional profile is performed on a samplederived from subjects.

The sample can be a sample selected from the group consisting of wholeblood, blood plasma, blood serum, red blood cells, urine, or tissuebiopsies. Depending on the determination method different samples willbe obtained from the same subject. It is also possible to use acombination of samples from the same subject. It is also contemplatedthat a sample or a set of samples is obtained from a subject at a firsttime point and at a second or further time point an additional sample orset of samples is obtained. In this way, it is possible to measurechanges of the nutritional profile in the subject over a time period.

Samples are processed according to technical requirements of subsequentanalyses.

For instance, biological samples can be directly injected, afterdilution, into the inductively coupled mass spectrometer for mineral andtrace element analysis (elemental analysis including analysis ofminerals). They can also be submitted to various steps of proteinprecipitation, extraction, clean-up, derivatization before beinginjected either on a gas liquid chromatographic system coupled to aflame ionization detector for fatty acid analysis or on a high pressureliquid chromatography coupled to mass spectrometry for the analysis ofamino acids or hydrosoluble vitamins. Biological samples can also beprocessed to undergo colorimetric, fluorometric or immuno-assayanalyses.

Nutritional profile and markers to be determined:

The nutritional profile encompasses direct and indirect markers. Themarkers can be biochemical, biological, or functional markers, or acombination thereof. Those markers are relevant for the nutritionalstatus of a subject and thus indicate their nutritional requirementswith respect to nutrients. These markers can be influenced by thenutrition consumed by the subject. These markers can be determined bythe various methods described below.

The quantitative analysis of these markers indicate the nutritionalrequirements for nutrients. Nutrients can be macronutrients andmicronutrients. Macronutrients can be protein, peptides, amino acids,fat, fatty acids, carbohydrates or choline. Micronutrients can beminerals, vitamins, carotenoids, phytonutrients. The nutrients can alsobe grouped by their function (functional nutrients), not depending ontheir structural similarities. For example, antioxidants provide anantioxidative effect, not dependent on their structure. Antioxidants canbe vitamins, minerals, amino acids or peptides.

The status of at least 10, 25, 50, 100, 250 markers can be determined.There is no particular requirement for a maximum number of to bedetermined markers but an upper limit might be 25, 50, 100, 250, or 1000markers.

Protein Markers:

Protein markers can be determined by markers of protein status ormarkers of protein catabolism and thus the nutritional requirements fornutrients providing protein or amino acids, or particular proteins andamino acids, can be determined. Protein status markers can be selectedfrom the group consisting of albumin, pre-albumin, or/andphosphocreatine and combinations thereof. Markers of catabolism areselected from the group consisting of ammonia, urea, modified aminoacids (monomethyl and dimethylarginine) or a combination thereof.

Amino Acid Markers:

Amino acid markers can be determined by determining the quantitation ofamino acids or their derivatives, including theirmetabolites/catabolites, in a sample as an indication of their statusand thus the nutritional requirements for nutrients providing protein oramino acids, or particular proteins and amino acids, can be determined.

Amino acid markers selected from the group consisting of alanine,β-alanine, sarcosine, arginine, monomethylarginine,assymetric-dimethylarginine, symmetric dimethylarginine, asparagine,aspartic acid, citrulline, glutamic acid, glutamine, glycine, histidine,1-methylhistidine, 3-methylhistidine, isoleucine, leucine, lysine,methionine, ornithine, phenylalanine, proline, serine, taurine,threonine, tryptophan, tyrosine, valine, hydroxyproline, ethanolamine,α-aminobutyric acid, β-aminoisobutyric acid, γ-aminobutyric acid,homocysteine, cysteine, γ-glutamyl-cysteine, cysteinyl-glycine,homocystine, cysteine, cystathionine, methionine sulfoxid,selenomethionine, methionine sulfoximine, selenocysteine, selenocystine,ergothioneine, N-formyl-L-methionine, S-adenosylhomocysteine,S-Adenosylmethioninamine, alpha-ketobutyric acid, 2-aminobutyric acid,2-amino-3-ketobutyric acid, alpha-keto-beta-methylbutyric acid (oralpha-ketoisovaleric acid), alpha-ketoisocaproic acid, oralpha-keto-beta-methylvaleric acid.

According to a preferred embodiment, the status of threonine, serine, orproline, preferably threonine, and thus the requirement thereof, isdetermined.

Preferably, the indicator of the status of threonine is threonine and/orone catabolite or several catabolites of threonine. The catabolite(s) ofthreonine is(are) selected from the group consisting of propionic acid,2-aminobutyric acid, 2-ketobutyric acid, 2-amino-3-ketobutyric acid,aminoacetone, acetylCoA, glycine, or a combination thereof (FIG. 3)

Preferably, the indicator of the status of isoleucine is isoleucine or acatabolite of isoleucine. The catabolite of isoleucine to be determinedis preferably 2-keto-3-methyl valeric acid (FIG. 3).

Fatty Acid Markers:

Fatty acid markers can be determined by the quantitative analysis offatty acids indicating their relative status and thus the nutritionalrequirements for nutrients providing fat, phospholipids(phosphatidylcholine, phosphatidylserine, phosphatidyletanolamine, etc.)or particular fatty acids can be determined.

Fatty acid markers selected from the group consisting of butyric C4:0,caproic C6:0, caprilic C8:0, capric C10:0, undecanoic 011:0, lauricC12:0, tridecanoic C13:0, myristic C14:0, pentadecanoic C15:0, palmiticC16:0, heptadecanoic C17:0, stearic C18:0, arachidic C20:0,heneicosanoic C21:0, behenic C22:0, lignoceric C24:0, myristoleic C14:1n-5, cis-10-Pentadecenoic C15:1 n-5, palmitoleic C16:1 n-7,cis-10-heptadecenoic C17:1 n-7, elaidic C18:1 n-9 trans, oleic C18:1 n-9cis, cis-11-Eicosenoic C20:1 n-9, erucic C22:1 n-9, nervonic 024:1 n-9,linoelaidic C18:2 n-6 trans, linoleic C18:2 n-6 cis, gamma-linolenicC18:3 n-6, alpha-Linolenic C18:3 n-3, cis-11,14-Eicosadienoic C20:2 n-6,cis-8,11,14-eicosatrienoic C20:3 n-6, cis-11,14,17-eicosatrienoic 20:3n-3, arachidonic C20:4 n-6, cis-13,16-docosadienoic 22:2 n-6,cis-5,8,11,14,17-eicosapentanoic (EPA) C20:5 n-3,cis-4,7,10,13,16,19-Docosahexaenoic (DHA) C22:6 n-3 acid, or lipoicacid.

Element Markers (Including Mineral Markers):

Direct Element (including mineral) markers are measured in a sampleeither as their quantitation through elemental analysis and/or using thequantitation of their associated proteins or metabolites such asferritin for iron status, ceruloplasmin for copper status, etc.

Element markers (including mineral markers) can be selected from thegroup consisting of lithium (Li), boron (B), magnesium (Mg), aluminium(Al), silicon (Si), phosphorous (P), sulfur (S), potassium (K), calcium(Ca), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt(Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se),bromine (Br), rubidium (Rb), strontium (Sr), molybdenum (Mo), tin (Sn),iodine (I), barium (Ba), titanium (Ti), sodium (Na), chlorine (Cl),fluorine (F).

Indirect element markers (including mineral markers) encompasscombination of one or several elements and/or their associated proteinsor metabolites.

Direct and indirect markers can be used to determine the dietaryrequirements for elements (including minerals).

Vitamin Markers:

Vitamin markers can be either direct or indirect markers. Direct markersencompass the quantification of vitamins and/or their metabolic productsin a sample. Indirect markers encompass combination or one or severalvitamins and/or their metabolic products as well as functional markersindicative of the status of vitamins such as for instance erythrocytetransketolase activity, erythrocyte glutathione reductase activity forthe status of vitamin B1 (thiamine) and vitamin B2 (riboflavin),respectively.

Vitamin markers can be used to determine the dietary requirements forvitamins. The vitamins markers can be selected from the group consistingof hydrosoluble vitamins or liposoluble vitamins.

The hydrosoluble vitamins and their metabolites can be selected from thegroup consisting of vitamin B1 (thiamine), vitamin B2 (riboflavin),vitamin B3 (nicotinic acid or niacin), nicotinamide, methylnicotinamide,nicotinuric acid, choline, vitamin B5 (pantothenic acid), vitamin B6(pyridoxine, pyridoxal, pyridoxamine), pyridoxal phosphate, pyridoxicacid, vitamin B8 (biotin), vitamin B9 (folic acid), methyltetrahydrofolic acid, vitamin B12 (cyanocobalamin, methylcobalamin),hydroxycobalamin, adenosylcobalamin.

The liposoluble vitamins can be selected from the group consisting ofvitamin A (retinol), vitamin K2 (menaquinone), vitamin K1(phylloquinone), vitamin E (α-tocopherol, δ-tocopherol), vitamin D(25-OH vitamin D2, vitamin D25-OH D3, vitamin D2, vitamin D3,1α-25-(OH)2 vitamin D3).

Nucleotide Markers:

Nucleotide markers and thus the requirements for nutrients providingnucleotides can be determined.

The nucleotides can be selected from the group consisting of inosine5″monophosphate, adenosine 5″monophosphate, cytidine 5″monophosphate,guanosine 5″-monophosphate, inosine 5″monophosphate, uridine5″monophosphate, or combinations thereof.

Phytonutrient Markers:

Phytonutrient markers and thus the requirements for nutrients providingphytonutrients can be determined.

In an embodiment the phytonutrients can be selected from the groupconsisting of carotenoids (e.g. lutein, zeaxanthin), ellagic acid,flavonoids (catechin, epicatechin, epigallocatechin), chlorogenic acids,resveratrol, glucosinolates, phytoestrogens (genistein, daidzein, etc.),or a combination thereof.

Peptide Markers:

Peptide markers and thus the requirements for nutrients providingpeptides can be determined.

The peptides can be selected from the group consisting of reducedgluthatione or oxidized glutathione.

Oxidative Stress Markers:

Oxidative stress markers, and thus the requirements for nutrientsimproving oxidative stress status, can be determined.

In an embodiment the oxidative stress markers can be selected from thegroup consisting of 4-hydroxynonenal, malondialdehyde, nitrotyrosine,carbonylated proteins, total glutathione, reduced glutathione, oxidizedglutathione, glutathione peroxidase activity, glutathione reductaseactivity, superoxide dismutase activity, catalase activity, or acombination thereof.

Nutrients affecting the oxidative stress status can be dietary oxidants.The dietary oxidants can be vitamins, minerals (e.g. selenium),phytochemicals, amino acids (e.g. cysteine) or peptides (e.g.glutathione). These nutrients reestablish oxidative stress level similarto the one of healthy subject.

Nitric Oxide Synthase Activity: Markers of nitric synthase activity andthus the requirements for nutrients affecting nitric oxide synthaseactivity can be determined.

In an embodiment, markers of nitric synthase activity can be selectedfrom the group consisting of nitrite, nitrate, monomethyl arginine,asymmetric dimethylarginine, symmetric dimethylarginine, arginine,citrulline, ornithine, argininosuccinic acid or a combination thereof.

Osmolyte Markers:

Markers for osmolytes and thus the requirements for nutrients affectingosmolyte status can be determined. These markers are functional markers.

In an embodiment, the osmolyte markers can be selected from the groupconsisting of trimethylamine N-oxide, dimethylsulfoniopropionate,trimethylglycine, sarcosine, betaine, glycerophosphorylcholine,myo-inositol, or a combination thereof.

Methods for Quantifying Markers:

Various methods are known for determining the above described markers ofnutritional status in a sample. Non-limiting examples of various methodsof how to perform a quantification of said markers, are described below.Other suitable methods known in the art may additionally/alternately beused.

The nutritional status of a subject in the sense of the inventionrelates to a profile of the status of direct or indirect markers, thatcan indicate the status of nutrients and micronutrients, which can bebiochemical, biological, functional, or other markers.

Direct biochemical markers can be the concentration of particularnutrients. Nutrients can be macronutrients (e.g. proteins and derivedamino acids, carbohydrates, lipids) or micronutrients (vitamins andelements (including minerals)).

An indirect biochemical marker can be a nutrient status indicator thateither relates to macronutrients or micronutrients, or a combinationthereof.

A nutrient status indicator can be a biomarker whose concentrationindicates the status of a nutrient without being said nutrient itself.For example, ferritin for iron or 25-hydroxy-vitamin D vitamers forvitamin D.

Other nutrient status markers can be derived from concentrations ofsingle nutrients or its metabolites or combination of concentrationsfrom nutrients and their metabolites (ratios) or combination ofconcentrations of nutrients with other biochemical markers (e.g.transport protein for nutrients/micronutrients) and/or biological and/orfunctional markers (e.g. specific enzymatic activity such astransketolase activity, erythrocyte glutathione reductase activity).

Various methods exist to determine the status of direct or indirectbiochemical markers. In the following we will discuss variousquantification methods for said markers. However, it should beunderstood that the respective method of quantification is not decisivefor the method of the invention as long as it outputs the nutritionalrequirement for a particular nutrient or set of nutrients.

Indicator of Amino Acid Oxidation (IAAO) Methodology:

As an example, the specific requirement for an essential amino acid canbe quantified using the indicator of amino acid oxidation (IAAO)methodology (Roberts S A, Thorpe J M, Ball R O, Pencharz P B.; Am J ClinNutr. 2001 February, 73(2):276-82.; Elango R, Ball R O, Pencharz P B., JNutr. 2008 February; 138(2):243-6. Review). Because of the limitationswith N-balance the stable isotope carbon oxidation based methods havebeen developed to evaluate essential amino acid requirements in humans(Pencharz P B, Ball R O. Different approaches to define individual aminoacid requirements. Annu Rev Nutr. 2003; 23:101-16.). The IAAO method hasbeen validated for estimating amino acid requirements with minimal prioradaptation (Bross R, Ball R O, Pencharz P B., J Nutr. 1998 November;128(11):1913-9; Thorpe J M, Roberts S A, Ball R O, Pencharz P B., JNutr. 1999 February; 129(2):343-8.). The IAAO technique is based on theconcept that when one essential amino acid is deficient for proteinsynthesis, then all other amino acids including the so-called indicatoramino acid (usually L-[1-¹³C]phenylalanine) are in excess since lessused, and therefore will be oxidized (Pencharz and Ball 2003). This isprimarily because excess amino acids cannot be stored and therefore mustbe partitioned between incorporation into protein or oxidation. Withincreasing intake of the limiting amino acid, oxidation of the indicatoramino acid will decrease, reflecting increasing incorporation intoprotein. Once the requirement is met for the limiting amino acid, therewill be no further change in the oxidation of the indicator amino acidwith increasing intake of the test amino acid. The inflection pointwhere the oxidation of the indicator amino acid stops decreasing andreaches a plateau is referred to as the ‘breakpoint’. The breakpoint,identified with the use of bi-phase linear regression analysis,indicates the estimated average requirement of the limiting (test) aminoacid. A particular strength of the IAAO model is that the absolute levelof oxidation does not matter, rather the relative oxidation across thebroad range of intake levels result in the same break-point (requirementestimate). This method is a well-accepted approach but it presents majorlimitations. Indeed it can only be applied to essential amino acids, itallows the assessment of only one essential amino acid per clinicalstudy, it is invasive for the patient (kinetic studies of tracers andseveral time-points with different diets), and it is time consuming.

Nutritional Profile (or Nutrient Profiling):

Nutrient requirements can be determined using the quantitative analysisof nutrients and their metabolic products, i.e. referred as nutrientprofiling, in biological samples. Nutrient profiling is achieved using acombination of analytical methods based on analytical techniques such ashigh performance liquid chromatography, gas chromatography, massspectrometry, spectrophotometry or immuno assays. Nutrient profilingencompasses concentration determination of a broad range of nutrientsand micronutrients and their metabolic products (metabolites) as well asrelative protein nutrient/micronutrient transporters, or functionalbiomarkers such as nutrient/micronutrient specific enzymatic activities.This nutrient profiling approach has the advantage to cover acomprehensive range of nutrients and micronutrients, enabling thus thepossibility to assess nutrient-nutrient interactions, as well as to befaster and relatively less invasive for the patients as no kineticstudies are needed.

Related to the determination of amino acid requirements, the concomitantanalysis of concentrations of amino acids and their specific metabolicproducts can be used to determine specific amino acid oxidation. Themeasurement of oxidation of specific amino acids, as a result of proteinoxidation, can be used to infer their relative incorporation intoproteins and therefore their specific requirements to meet the metabolicdemand for protein synthesis. For instance, threonine is oxidized into2-keto butyric acid 2-aminobutyric acid, and 2-amino-3-ketobutyric acid(FIG. 3).

As an example, the concomitant analysis of the concentrations ofthreonine and its metabolites can be used to assess threonine oxidation(see FIG. 3, left side). The threonine oxidation is then compared to theone from another essential amino acid, taken as benchmark, such as forinstance isoleucine (see FIG. 3, right side). In the case of isoleucine,the oxidation is measured using determination of concentrations ofisoleucine and its oxidation product, 2-keto 3-methylvaleric acid (seeFIG. 2, right side). Oxidation is defined as the calculated product ofthe combination of concentrations of threonine and its metabolite(s),such as:

-   -   Ratio concentration of L-threonine/concentration of 2-keto        butyric acid    -   Ratio concentration of L-threonine/(concentration of 2-keto        butyric acid+concentration of 2-aminobutyric acid)    -   Ratio concentration of L-threonine/(concentration of 2-keto        butyric acid+concentration of 2-aminobutyric acid+concentration        of 2-amino 3-ketobutyric acid)    -   or any mathematical combination of thereof    -   or any mathematical combination of thereof and other markers or        product of protein catabolism such as concentration of        circulating ammonia, intermediates in urea cycle (ornithine,        citrulline, arginine succinic acid, arginine), concentrations of        symmetric dimethylarginine, asymmetric dimethylarginine.    -   Any combination of thereof and markers of oxidative stress,        nitric oxide metabolism (nitric oxide levels in blood        plasma/serum, nitrate levels in urine).    -   Any combination of thereof and clinical markers used to monitor,        for example, IBD activity: CRP, differential blood count, fecal        calprotectin, iron status, blood sedimentation rate, protein        electrophoresis, fecal neutrophils, vitamin B12 status, and        others.

For example, the oxidation of threonine and isoleucine can be comparedbetween a diseased and a healthy subject as has been done in FIG. 4(here the example relates to IBD as the disease of interest). As canbeen seen from FIG. 4 only in the case where the threonine oxidation islower compared to the healthy subject an enhanced nutritionalrequirement for threonine in IBD can be identified.

As such the invention also relates to a method for determining thenutritional requirements for threonine and/or isoleucine comprisingcomparing the oxidation of threonine and/or isoleucine of diseased and ahealthy subject.

Amino acid quantification with Ultra Performance Liquid Chromatographycoupled to tandem mass spectrometry:

Amino acids can be also directly measured in a sample obtained from thesubject.

Sample Preparation

50 μL of blood plasma or serum is added with 10 μL labelled internalstandards and 140 μL of cold methanol (0.1% formic acid) for proteinprecipitation. Samples then undergo agitation with vortex (5 min)followed by centrifugation at 10000 rpm for 10 min at 4° C. Supernatantis then collected to undergo derivatization.

Amino Acid Derivatization

Derivatization is performed using the AccQ-Tag Ultra Derivatization KitAmino Acid Analysis (Waters Corp.) following manufacturer's procedures:10 μL of either a standard amino acid mix solution or the supernatant ofthe sample is mixed with 70 μL of AccQ-Tag Ultra borate buffer (pH=8.8).The derivatization is carried out by adding 20 μL of reconstitutedAccQ-Tag Ultra reagent (3 mg/mL of 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate, or AQC in acetonitrile) to the buffered mixture. The sampleis then immediately vortexed followed by incubation for 15 min at 55° C.

Analysis of Amino Acids Using Ultra Performance Liquid ChromatographyCoupled to Tandem Mass Spectrometry (UPLC-MS/MS)

UPLC-MS/MS analysis is performed on a Waters Acquity UPLC system on-linecoupled to a Waters Xevo TQ mass spectrometer by means of anelectrospray ionization (ESI) probe. Chromatographic separation isachieved using a Waters AccQ-Tag Ultra column (2.1 mm i.d.×100 mm, 1.7μm particles) using a binary system of eluents A and B. Eluent Acontains 10% of commercial solution of AccQ-Tag Ultra Eluent Aconcentrate (Waters Corp.) in 90% water. Eluent B is a commercialsolution of AccQ-Tag Ultra Eluent B (Waters Corp.). The separationgradient used is: 0-0.54 min (99.9% A), 5.74 min (90.9% A), 7.74 min(78.8% A), 8.04 (40.4% A), 8.05-8.64 (10% A), 8.73-10.0 (99.9% A). Theautosampler temperature is set at 20° C. and the column temperature at55° C. The sample injection volume is 2 μL. Cone voltage and collisionenergy values are determined on each measured amino acid using theWaters IntelliStart routine. The ion m/z 171 representing the commonmain product from the collision-induced dissociation of all the AQCadducts, was used for the quantification of individual amino acids.Retention times of amino acids are determined using injection ofstandard amino acid solutions into the UPLC-ESI-MS/MS system. Thefollowing ionization source settings were used: capillary voltage, 2.5kV (ESI+); desolvation temperature, 600° C.; desolvation gas flow rate,1000 L/h; source temperature, 150° C.

The analyzer settings are determined during each calibration periodswith typical values as following: first quadrupole 2.95 (low massresolution), 14.35 (high mass resolution), ion energy 1:0.1; secondquadrupole 2.95 (low mass resolution) and 14.40 (high mass resolution),ion energy 2:0.3. Argon was used as collision gas at a flow rate of 0.15mL·min⁻¹. The UPLC-MS/MS system control and data acquisition wereperformed with the Waters Corporation MassLynx™ software. Data analysiswas conducted with the TargetLynx™ software (Waters Corporation).

Quantification of Fatty Acids by Gas-Liquid Chromatography:

The concentration of fatty acids can be determined with any suitablemethod like thin layer chromatograph or gas chromatography.

In the following we will exemplify a gas chromatography method.

Sample Preparation

Derivatization of plasma and red blood cell (RBC) fatty acids areperformed under acidic conditions. Briefly, 200 μL of sample are mixedwith methanol, methanolic hydrochloric acid, hexane and internalstandard solution in screw-capped glass tubes. The tubes are capped andheated at 100° C. for 60 min for plasma and 90 min for RBC followed by acooling phase to room temperature and an addition of water to stop thereaction. Then the tubes are centrifuged at 1200 g for 5 min and theupper organic phase is collected and analyzed by gas chromatography(GC).

Fast Analysis of Fatty Acid Methyl Esters (FAMEs) by Gas-LiquidChromatography

Analysis of total FAM Es is performed on a 7890 Agilent gaschromatograph (Agilent Technologies, Palo Alto, Calif., USA), equippedwith a fused-silica BPX-70 capillary column (10 m×0.1 mm I.D., 0.2 mfilm thickness; SGE, Melbourne, Australia). Split injector (35:1) andflame-ionization detection (FID) system were operating at 250° C. and300° C. respectively. The volume of the oven has been reduced to about5400 cm³ using a commercial device obtained from Agilent. Oventemperature programming was 45° C. isothermal for 0.5 min, increased to180° C. at 100° C./min, isothermal for 0.5 min at this temperature thenincreased to 220° C. at 9° C./min, isothermal for 0.5 min at thistemperature and then to 250° C. at 50° C./min (total run time 7.9 min).The carrier gas (H₂) flow was maintained constant at 0.7 mL·min⁻¹ andthe acquisition of the FID signal at 50 Hz.

Identification of FAMEs

A mixture of standard FAM Es is used to confirm identification of fattyacids. The mixture contains methyl esters of: butyric acid (4:0),caproic acid (6:0), caprylic acid (8:0), capric acid (10:0), undecanoicacid (11:0), lauric acid (12:0), tridecanoic acid (13:0), myristic acid(14:0), myristoleic acid (14:1 n-5), pentadecanoic acid (15:0),pentadecenoic acid (15:1 n-5), palmitic acid (16:0), palmitoleic acid(16:1 n-7), heptadecanoic acid (17:0), heptadecenoic acid (17:1 n-7),stearic acid (18:0), elaidic acid (trans-18:1 n-9), oleic acid (18:1n-9), linolelaidic acid (all trans-18:2 n-6), linoleic acid (18:2 n-6),arachidic acid (20:0), α-linoleic acid (18:3 n-6), eicosenoic acid (20:1n-9), linolenic acid (18:3 n-3), heneicosanoic acid (21:0),eicosadienoic acid (20:2 n-6), behenic acid (22:0), eicosatrienoic acid(20:3 n-6), erucic acid (22:1 n-9), eicosatrienoic acid (20:3 n-3),arachidonic acid (20:4 n-6), docosadienoic acid (22:2 n-6), lignocericacid (24:0), eicosapentanoic acid (20:5 n-3), nervonic acid (24:1 n-9)and docosahexaenoic acid (22:6 n-3).

Quantification of Sulfur Containing Molecules:

Sulfur containing molecules encompass amino acids and derivates(homocysteine, cysteine, α-glutamyl-cysteine, cysteinyl-glycine,homocystine, cysteine, cystathionine, methionine sulfoxid,selenomethionine, methionine sulfoximine, selenocysteine, selenocystine,ergothioneine, N-formyl-L-methionine, S-adenosylhomocysteine, S-Adenosylmethioninamine), peptides (reduced gluthatione and oxidizedgluthatione), and lipoic acid.

Sample Preparation

Blood plasma, serum or red blood cell samples (50 μl) are mixed with 50μl of internal standard glutathione ethyl ester (GSHee) before beingtreated with 100 μl of derivatization solution containing 10 mMlodoacetic acid in 10 mM aqueous ammonium bicarbonate and ammoniac (0.5%v/v, pH 9.5). This mixture is stored at room temperature for 15 minutes.The reaction is stopped and the proteins are precipitated by addition of50 μl of cold sulfosalicylic acid solution (10% w/v). The mixture isthen centriguged at 16000×g at 4° C. for 15 minutes. The supernatant(200 μl) is transferred to glass vials and 2 μl were injected into the

Analysis of Derivatized Sulfur Containing Molecules Using UltraPerformance Liquid Chromatography Coupled to Tandem Mass Spectrometry(UPLC-MS/MS)

UPLC-MS/MS analysis is performed on a Waters Acquity UPLC system on-linecoupled to a Waters Xevo TQ mass spectrometer by means of anelectrospray ionization (ESI) probe. Chromatographic separation isachieved using a Waters HSS T3 2.1 mm+100 mm, 1.7 μm column. Elution isperformed at a flow rate of 0.25 mL/min using a gradient composed ofsolvents A (0.1% formic acid in water) and B (acetonitrile/water 20:80,v/v with 0.1% formic acid). The gradient was as follows: 100% solvent A0-2 min, 1% solvent A 2-7 min, 99% solvent A 7.1-10 min. The massspectrometer operates under the following conditions: capillary voltage:2.5 KV; source temperature: 150° C.; desolvation temperature: 600° C.;desolvation gas flow: 1000 L/Hr. For quadrupole 1, the low and high massresolution are 2.95 and 14.35, respectively, with a ion energy of 0.1.For the quadrupole 2, the low and high mass resolution are 2.95 and14.40, respectively, with a ion energy of 0.3. Argon is used ascollision gas at a flow rate of 0.15 mL/min. MRM transitions with theirrespectively optimized cone voltage and collision energy values weredetermined for each metabolite using the Waters IntelliStart software.Using these conditions, each sulfur containing molecules is injectedinto the UPLC-MS/MS system to determine the retention time. The MRM-MSmethod is built to monitor only one transition channel per MRM function.The UPLC-ESI-MS/MS system control and data acquisition were performedwith the Waters Corporation MassLynx™ software. Data analysis wasconducted with the TargetLynx™ software (Waters Corporation).

Quantification of Elements (Including Minerals):

Elements (including minerals) can be quantified by any suitable methodknown in the art. An example is provided in the following.

Sample Preparation

A volume 150 μL of biological fluid (blood plasma, serum, urine, etc.)is diluted 1:10 in a diluent solution containing 5% 1-butanol, 0.05%EDTA, 0.05% Trition X-100, and 1% ammonium hydroxid.

Elemental Analysis Using Inductively Coupled Plasma Triple QuadrupoleMass Spectrometry (ICP-MS/MS)

Elemental analysis is performed using a 8800 ICP-MS/MS spectrometer(Agilent Technologies, Tokyo, Japan) that is operated in low matrixplasma mode. The ICP-MS system is equipped with an integrated autosampler for direct sample introduction. The samples are pumped at a flowrate of 0.35 mL min⁻¹ through an integrated peristaltic pump into thesample introduction area, consisting of a concentric nebulizer and aScott double-pass spray chamber. After ionization in the plasma, theanalyte-ions are transferred into the mass spectrometer. The ICP-MSsystem is equipped with a triple-quadrupole mass analyzer which allowsapplying MS/MS analysis mode. Quantification of each mineral is achievedby external calibration. In order to correct or reduce plasmafluctuations and matrix effects, on-line dilution is applied to mix thesamples with a multi-element internal standard solution. For qualitycontrol, a human certified reference material (Seronorm Trace ElementsSerum L-1 from Sero, Norway) is analysed during each analytical run. Themeasured elements are listed on Appendix 1.

Element Speciation Analysis Using High Performance Liquid ChromatographyCoupled to Inductively Coupled Plasma Triple Quadrupole MassSpectrometry (UHPLC-ICP-MS/MS)

For all chromatographic experiments, a 1290 Infinity dual-piston UHPLCpump (Agilent Technologies, Tokio, Japan) is used. Analysis of mineralspecies, weakly associated to biomolecules, is achieved by sizeexclusion chromatography coupled to ICP-MS, applying 50 mM ammoniumacetate (pH 7.4) as mobile phase in isocratic elution mode. For theanalysis of stable species, reversed phase (RP) HPLC-ICP-MS is used,applying gradient elution at a flow rate of 0.4 mL min⁻¹. Mobile phase Ais composed of 2% acetonitrile (ACN), 0.05% TFA (trifluoroacetic acid)and mobile phase B of 98% ACN, 0.05% TFA. A typical gradient consists ofa linear increase of acetonitrile from 5% to 80% within 30 min. Foranalysis of biological fluids an injection volume of 3-20 μL is applied.Identification of the mineral species is achieved by collectingfractions corresponding to the detected chromatographic peaks. Finally,the collected fractions were preconcentrated, desalted and analysed bymolecular MS for identification of the biomolecules. Quantification isachieved by post-column isotope dilution analysis. Here, theisotopically enriched elements are dissolved in 2% nitric acid andinjected by a peristaltic pump, maintaining a constant flow rate of 0.2mL min⁻¹.

Hydrosoluble Vitamins:

Hydrosoluble vitamins can be directly quantified in a sample obtainedfrom the subject.

Chemicals

Vitamin standards, formic acid, acetic acid are purchased fromSigma-Aldrich (St. Louis, Mo.), Millipore water were from Water(Milford, Mass.), acetonitrile was purchased from VWR (Radnor, Pa.).Labelled vitamin standards are purchased from Cambridge IsotopeLaboratories (Tewksbury, Mass.).

Sample Preparation

Plasma/serum was stored at −80° C. unfrozen and immediately put on ice.Plasma/serum is spiked with labeled internal standards mixed and keptfor 10 min. Proteins are precipitated by mixing 100 μl of plasma/serumwith 100 μl of 10% TCA acid. Samples are extracted for 10 min on ice andcentrifuged at 4° C. for 10 min at 17000 rpm for the analysis of B1, B2,B3, B6, B7 vitamins and metabolites. 100 μl of serum/plasma is mixedwith 200 μl 90% methanol/water solution containing acetic and ascorbicacids for the analysis of vitamin B9. Samples are stirred for 20 min,centrifuged and supernatant was dried down under nitrogen stream.Samples were re-suspended with water and injected using describedearlier UPLC conditions.

Analysis of B1, B2, B3, B6, B7 and B9 Vitamins and Metabolites UsingUltra Performance Liquid Chromatography coupled to tandem massspectrometry (UPLC-MS/MS)

10 μl of supernatant is injected into a Waters Acquity UPLC systemon-line coupled to a Waters Xevo TQ mass spectrometer by means ofelectrospray ionization (ESI) probe. Chromatographic separation isachieved using a Waters Acquity UPLC® HSS T3 2.1×100 mm column, 0.6 mlflow rate, solvent A—0.1% formic acid in water, solvent B—0.1% formicacid in acetonitrile. Gradient program from 100% of A to 100% of Bwithin 9 min. Detection of vitamins was performed on Waters XEVO TQSmass spectrometer in scheduled MRM mode with 2 transitions per vitamin.The UPLC-MS/MS system control and data acquisition were performed withthe Waters Corporation MassLynx™ software. Data analysis was conductedwith the TargetLynx™ software (Waters Corporation).

Liposoluble Vitamins:

Liposoluble vitamins can be directly quantified in a sample obtainedfrom the subject.

Chemicals

Hexane, methanol, ethanol, deionized water and acetonitrile arepurchased from VWR (Radnor, PE) international. Standards of vitamins arepurchased from Sigma Aldrich (St. Louis, Mo.). Analytical column (HSSC18, 1.7□m 3×100 mm) and solid-phase extraction (SPE) plats arepurchased from Waters (Milford, Mass.).

Sample Preparation

In order to protect vitamins from light degradation, the samplepreparation steps are performed under ultraviolet (UV) protection bymeans of filters properly implemented in the laboratory.

Liposoluble vitamins are extracted using a protein precipitationcombined with a liquid-liquid extraction procedure. Briefly, 200 □l of asolution of butylated hydroxyl toluene (BHT) in ethanol are added to 200□l of human serum to perform protein precipitation and samplepreservation from oxidation. Samples are then extracted with 2.5 ml ofn-hexane, sonicated and centrifuged for three times. The collectedsupernatants are combined and dried under nitrogen flow to be finallyreconstituted in n-hexane/isopropanol 9:1.

The two 25-hydroxyvitaminD metabolites undergo a different extractionprotocol. Briefly, 150 □l of human serum are added with using 150□□l ofmethanol) for protein precipitation. Supernatant fractions collected bycentrifugation (15 min at 4 C° 4000 rpm) are collected and submitted toSPE clean-up step using Oasis HLB (Waters, Milford, Mass., USA)cartridges preconditioned with methanol and water. Cartridges are loadedwith samples and washed with water and 5% methanol solution. The elutionof the analyses is obtained using methanol. The samples are then driedand reconstitute in n-hexane/isopropanol 9:1.

Analysis of Liposoluble Vitamins and Metabolites Ultra-PerformanceConvergence Chromatography to Tandem Mass Spectrometry (UPC²-MS/MS)

UPC²-MS/MS analysis is performed on a Waters Acquity Ultra-PerformanceConvergence Chromatography (UPC²) system equipped with Xevo TQS massspectrometer (Waters Corporation). An HSS C18 analytical column isutilized to perform the chromatographic separation and it connected withpumps: A (mobile phase, CO2) and Pump B (mobile phase, 10 mM ammoniumacetate Methanol solution). The gradient applied starts from 2% oforganic solvent until 40% in a total run time of 14 minutes using 1.2 mlflow rate. The MS analysis are as follows: capillary voltage: 2.6 kV,desolvation temperature: 500° C., cone gas flow rate: 150 L/hr,desolvation gas flow rate: 500 L/hr. The multiple reaction monitoring(MRM) is applied to perform this analysis. The UPLC-MS/MS system controland data acquisition were performed with the Waters CorporationMassLynx™ software. Data analysis was conducted with the TargetLynx™software (Waters Corporation).

For example, the amount of 25-hydroxy-vitamin D vitamins can be used todetermine the status of vitamin D.

Further Micronutrient Analyses:

Vitamin or mineral/trace element status can be performed using theexemplary methods described below.

Vitamin B12 and vitamin B9 (folate) status can be measured using thecompetitive principle.

The quantification of plasma/serum methylmalonic acid and totalhomocysteine, as well as plasma concentration of holotranscobalamin IIcan be performed for vitamin B12 status.

The quantification of ferritin, soluble transferrin transporter, orhepcidin can be performed for iron status.

The quantification of tyroxine T4 and T3 can be performed for iodinestatus.

The quantification of ceruloplasmin and copper/zinc superoxide dismutasecan be performed for copper status.

Further Analyses of Metabolites:

Additional analyses are performed on blood plasma/serum for ammonia andurea quantitation using a Cobas® C111 (Roche Diagnostics). Measured areperformed by spectrophotometry. Ammonia concentration is calculatedendpoint decrease at 340 nm (wavelength A) and 629 nm (wavelength B).Urea concentration is determined using a calculation mode by kineticdecrease at 340 nm (wavelength A) and 409 nm (wavelength B).

Compositions:

The compositions of the invention comprise nutrients in an amount thatreestablish in the diseased person the metabolic, physiologic andfunctional equivalence of the nutritional profile or status of a healthyperson or healthy cohort.

First, the distinctive nutritional requirements of the diseased personor a cohort of diseased patients with the same disease or the sameseverity grade of the disease are determined.

In this way, nutrients can be identified that are not provided inoptimal amounts to the diseased patient, thus are provided in a too lowor too high amount, generally in a too low amount.

Nutritional compositions can then be adapted to contain the nutrients inan amount that will recover the nutritional profile of the diseasedperson towards the nutritional profile of a healthy person. Optimally,the nutritional profile of the diseased subject will have the same oralmost identical nutritional status after consumption of the nutritionalcomposition adapted to the diseased subject.

For example, a nutritional composition for a diseased subject exhibitinga deficiency in threonine as determined by the IAAO methodology containsthreonine either in the form of free threonine or/and protein boundthreonine in an amount that will reduce or eliminate the threoninedeficiency.

Accordingly, the nutritional composition can either be a supplementcomprising only those nutrients that are underrepresented in a regulardiet.

Alternatively, the nutritional composition can be in the form of acomplete food providing all the nutrients that the diseased personrequires for normal nutritional status, and thus both nutrients forwhich the subject has distinctive nutritional requirements compared to ahealthy subject and those nutrients for which the subject has the samenutritional requirements as a healthy subject. A complete food mightcontain those nutrients in a less amount for which the diseased personhas a reduced requirement compared to a healthy subject.

Formulations:

The above described compositions can be formulated in liquid or solidform.

The compositions can further comprise at least one additional activeagent, carrier, vehicle, excipient, or auxiliary agent identifiable by aperson skilled in the art upon reading of the present disclosure.

The composition can be in the form of an a nutritional composition orpharmaceutical product. A nutritional composition or pharmaceuticalproduct can comprise the composition of the invention.

Nutritional Composition:

As used herein, the term “nutritional composition” includes, but is notlimited to, complete nutritional compositions, partial or incompletenutritional compositions, and disease or condition specific nutritionalcompositions. A complete nutritional composition (i.e., those whichcontain all the required essential macro and micro nutrients) can beused as a sole source of nutrition for the patient. Patients can receive100% of their nutritional requirements from such complete nutritionalcomposition. A partial or incomplete nutritional composition does notcontain all the essential macro and micro nutrients and cannot be usedas a sole source of nutrition for the patient. Partial or incompletenutritional compositions can be used as a nutritional supplement, i.e.in addition to a patient's diet. An oral supplemental nutritionalcomposition contains those nutrients for which the diseased person hasan increased demand compared to a healthy person as identified with themethod of the invention.

A complete nutritional composition typically has an energy density ofhaving a caloric density of 0.7-2.0 kcal/ml (2.9-8.4 kJ/ml).

A nutritional composition may comprise the following macronutrients andmicronutrients: a source of proteins, a source of lipids, a source ofcarbohydrates, vitamins and elements (including minerals).

The source of protein can be animal, milk, or plant protein.

The nutritional composition further includes one or more free aminoacids. Non-limiting examples of amino acids include alanine, arginine,asparagine, aspartate, citrulline, cysteine, glutamate, glutamine,glycine, histidine, hydroxyproline, hydroxyserine, hydroxytyrosine,hydroxylysine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, taurine, threonine, tryptophan, tyrosine, valine.Examples for non protein amino acids are citrulline, HICA(Alpha-Hydroxyisocaproic Acid), HIVA (Alpha-Hydroxyisovaleric Acid),HIMVA (alpha-hydroxymethylvaleric acid) or a combination thereof.

Free amino acids can be the only source of protein in the composition orcombined with other sources of protein. Each amino acid is present in anamount of 0.5%-25% of the total amino acids.

The source of fat may either be an animal fat or a vegetable fat orboth. Although animal fats have essentially equal caloric andnutritional values and can be used interchangeably, vegetable oils arepreferred in the practice of the present invention due to their readilyavailability, ease of formulation, and lower concentration of saturatedfatty acids. Fat sources to be used comprise fish oil, egg oil, algaloil, corn oil, sunflower oil, safflower oil, canola oil, coconut oiland/or soybean oil or combinations thereof.

The nutritional composition may comprise elements and minerals such asboron, calcium, calcium acetate, calcium gluconate, calcium chloride,calcium lactate, calcium phosphate, calcium sulfate, chloride, chromium,chromium chloride, chromium picolonate, copper, copper sulfate, coppergluconate, cupric sulfate, fluoride, iron, carbonyl iron, ferric iron,ferrous fumarate, ferric orthophosphate, iron trituration,polysaccharide iron, iodide, iodine, magnesium, magnesium carbonate,magnesium hydroxide, magnesium oxide, magnesium stearate, magnesiumsulfate, manganese, molybdenum, phosphorus, potassium, potassiumphosphate, potassium iodide, potassium chloride, potassium acetate,selenium, sulfur, sodium, docusate sodium, sodium chloride, sodiumselenate, sodium molybdate, zinc, zinc oxide, zinc sulfate and mixturesthereof. Non-limiting exemplary derivatives of mineral compounds includesalts, alkaline salts, esters and chelates of any mineral compound citedabove.

The nutritional composition may further comprise vitamins such asvitamin B1 (thiamin, thiamin pyrophosphate, TPP, thiamin triphosphate,TTP, thiamin hydrochloride, thiamin mononitrate), vitamin B2(riboflavin, flavin mononucleotide, FMN, flavin adenine dinucleotide,FAD, lactoflavin, ovoflavin), vitamin B3 (niacin, nicotinic acid,nicotinamide, niacinamide, nicotinamide adenine dinucleotide, NAD,nicotinic acid mononucleotide, NicMN, pyridine-3-carboxylic acid),vitamin B3-precursor tryptophan, vitamin B6 (pyridoxine, pyridoxal,pyridoxamine, pyridoxine hydrochloride), pantothenic acid (pantothenate,panthenol), folate (folic acid, folacin, pteroylglutamic acid), vitaminB12 (cobalamin, methylcobalamin, deoxyadenosylcobalamin, cyanocobalamin,hydroxycobalamin, adenosylcobalamin), biotin, vitamin C (ascorbic acid),vitamin A (retinol, retinyl acetate, retinyl palmitate, retinyl esterswith other long-chain fatty acids, retinal, retinoic acid, retinolesters), vitamin D (calciferol, cholecalciferol, vitamin D3,1,25,-dihydroxyvitamin D), vitamin E (a-tocopherol, a-tocopherolacetate, a-tocopherol succinate, a-tocopherol nicotinate, a-tocopherol),vitamin K (vitamin K1, phylloquinone, naphthoquinone, vitamin K2,menaquinone-7, vitamin K3, menaquinone-4, menadione, menaquinone-8,menaquinone-8H, menaquinone-9, menaquinone-9H, menaquinone-10,menaquinone-11, menaquinone-12, menaquinone-13), choline, inositol,6-carotene and any combinations thereof.

A complete nutritional composition typically comprises 10-40 en %protein, 10-60 en % carbohydrates, and 20-80 en % fat. “en %” is theamount of energy provided to the total of the energy of the nutritionalcomposition.

The composition may also contain anti-oxidants, stabilizers (whenprovided in solid form) or emulsifiers (when provided in liquid form).

Format of the Nutritional Composition:

In one embodiment, the nutritional composition is selected from thegroup consisting of supplemental nutritional composition, a completenutritional composition, a yoghurt product, fermented milk, a fruitjuice, a dried powder in sachet format or a cereal bar.

The nutritional composition may be a medical food, also referred to as afood for special medical proposes. A medical food product is speciallyformulated and intended for the dietary management of diseases ormedical conditions (e.g., prevent or treat diseases or undesirablemedical conditions). A medical food product can provide clinicalnutrition, for example fulfilling special nutritional needs of patientswith a medical condition or other persons with specific nutritionalneeds.

The medical food may be in the form of a health care nutritionalcomposition for oral feeding, and/or a nutritional product for enteralor parental feeding. In the case of a product for parenteral feeding itwill only include ingredients which are suitable for parenteral feeding.Ingredients that are suitable for parental feeding are known to theperson skilled in the art.

In an embodiment, the medical food can be in the form of a nutritionallycomplete product, a drink, a dietary supplement, a meal replacement, afood additive, a supplement to a food product, a powder for dissolution,an enteral nutrition product, an infant formula, and combinationsthereof.

In an embodiment, the nutritional composition may be in the form of afermented milk, a yogurt, a fresh cheese, a renneted milk, aconfectionery bar, breakfast cereal flakes, a breakfast cereal bar, adrink, a milk powder, a soy-based product, a non-milk fermented product,or a nutritional supplement for clinical nutrition. In an embodiment thecomposition may be in the form of a powder, in particular a powder forreconstitution with a liquid. In an embodiment the composition may be inthe form of a liquid, for example a ready-to-drink liquid oralnutritional supplement.

In an embodiment, the nutritional compositions are in a form selectedfrom the group consisting of tablets, capsules, liquids, chewables, softgels, sachets, powders, syrups, liquid suspensions, emulsions,solutions, or combinations thereof. In an embodiment, the nutritionalcompositions are oral nutritional supplements. Alternatively, thenutritional compositions may be tube feedings.

Viscosity:

If the nutritional composition is a liquid it has a viscosity below 150mPa·s, preferably below 100 mPa·s, more preferably below 80 mPa·s, evenmore preferably below 70 mPa·s. The viscosity is determined in arotational rheometer using a cone-plate geometry at 20° C. at a shearrate of 50 1/s.

If the composition is provided as texturised product (pudding etc.)ready for consumption to be eaten with a spoon a viscosity of at least350 mPa·s, preferably above 750 mPa·s, more preferably between 1000 and4000 mPa·s. is preferred.

Therapeutical Uses and Methods:

The composition of the invention can be used in the treatment orprevention of diseases or for methods for the treatment or prevention ofdiseases associated with a nutritional status in diseased subject thatdiffers from the nutritional status of a healthy subject.

In a preferred embodiment, the disease is an inflammatory bowel disease(e.g. Crohn's disease, ulcerative colitis, collagenous colitis,lymphocytic colitis, diversion colitis, Behçet's disease, indeterminatecolitis). Other diseases or clinical conditions for which the methodsand compositions of the invention are suitable include irritable bowelsyndrome, type 2 diabetes, Parkinson disease, Alzheimer disease,cognitive decline/impairment, depression, critical care conditions.

Methods of Production:

A method for producing the above described composition is provided andcomprises providing at least one of the above described nutrients, andadding optionally at least one further nutrient, said nutrients, forexample, selected from the group consisting of one or more amino acids,fat, or carbohydrate, optionally adding a carrier or/and water.

Those skilled in the art will understand that they can freely combineall features of the present invention disclosed herein. In particular,features described for different embodiments of the present inventionmay be combined. Further advantages and features of the presentinvention are apparent from the figures.

1-30. (canceled)
 31. A method for manufacturing a nutritionalcomposition for administration to a subject suffering from a diseasecharacterized by distinctive nutritional requirements in the subjectover a healthy subject, the method comprising: a. determining in asample of the subject suffering from the disease a profile of statusesof markers comprising direct and indirect markers indicating thenutritional profile of the subject, wherein each of the direct markersindicates an amount of a macronutrient or micronutrient, and each of theindirect markers are derived from the direct markers by quantifying acorresponding direct marker and quantifying one or more additionalmarkers that indicate a status of the corresponding direct marker, atleast one of the direct and indirect markers is selected from the groupconsisting of an amino acid marker, a fat marker, a carbohydrate marker,a nucleotide marker, an osmolyte marker, a peptide marker, a catabolismmarker, a nitric synthase marker, and combinations thereof; b.determining in a sample of the healthy subject a profile of the statusesof the same markers comprising the same direct and indirect markersdetermined in step a, at least one of the sample of the subjectsuffering from the disease or the sample of the healthy subject is asample selected from the group consisting of whole blood, blood plasma,blood serum, red blood cells, urine, tissue biopsies, and combinationsthereof; c. comparing the profiles determined in steps a. and b., anddetermining distinctive nutritional requirements for nutrientscomprising macronutrients and micronutrients in the subject sufferingfrom the disease based on the comparison of the profiles determined insteps a. and b., wherein when a level of the markers determined in stepa. is lower than a level of the same markers determined in step b., thedistinctive nutritional requirements for the subject comprise acomposition comprising the nutrients in an effective amount to enable alevel of the nutrients in the subject suffering from the disease tomatch a level of the nutrients in the healthy subject; and d.manufacturing the composition comprising the nutrients in the effectiveamount to restore the nutritional profile of the nutrients in thesubject suffering from the disease to or toward that of the healthysubject as determined in step b.
 32. The method of claim 31, wherein instep a., the statuses of the markers is determined in a cohort ofsubjects suffering from the disease.
 33. The method of claim 31, whereinin step b., the statuses of the markers is determined in a cohort ofhealthy subjects.
 34. The method of claim 31, wherein the statuses of atleast 10 markers are determined.
 35. The method of claim 31, wherein thecatabolism marker is selected from the group consisting of ammonia,urea, monomethyl, dimethylarginine, and combinations thereof.
 36. Themethod of claim 31, wherein the markers further comprise an oxidativestress marker selected from the group consisting of 4-hydroxynonenal,malondialdehyde, nitrotyrosine, carbonylated proteins, totalglutathione, reduced glutathione, oxidized glutathione, glutathioneperoxidase activity, glutathione reductase activity, superoxidedismutase activity, catalase activity, and combinations thereof.
 37. Themethod of claim 31, wherein the nitric synthase marker is selected fromthe group consisting of nitrite, nitrate, monomethyl arginine,asymmetric dimethylarginine, symmetric dimethylarginine, arginine,citrulline, ornithine, argininosuccinic acid, and combinations thereof.38. The method of claim 31, wherein the amino acid marker comprises amarker of an amino acid selected from the group consisting of aminoalanine, β-alanine, sarcosine, arginine, monomethylarginine,assymetric-dimethylarginine, symmetric dimethylarginine, asparagine,aspartic acid, citrulline, glutamic acid, glutamine, glycine, histidine,1-methylhistidine, 3-methylhistidine, isoleucine, leucine, lysine,methionine, ornithine, phenylalanine, proline, serine, taurine,threonine, tryptophan, tyrosine, valine, hydroxyproline, ethanolamine,α-aminobutyric acid, β-aminoisobutyric acid, γ-aminobutyric acid,homocysteine, cysteine, γ-glutamyl-cysteine, cysteinyl-glycine,homocystine, cysteine, cystathionine, methionine sulfoxide,selenomethionine, methionine sulfoximine, selenocysteine, selenocystine,ergothioneine, N-formyl-L-methionine, S-adenosylhomocysteine,S-Adenosylmethioninamine, alpha-ketobutyric acid, 2-aminobutyric acid,2-amino-3-ketobutyric acid, alpha-keto-beta-methylbutyric acid,alpha-ketoisovaleric acid, alpha-ketoisocaproic acid,alpha-keto-beta-methylvaleric acid, and combinations thereof.
 39. Themethod of claim 31, wherein the markers further comprise a fatty acidmarker selected from the group consisting of butyric C4:0, caproic C6:0,caprilic C8:0, capric C10:0, undecanoic C11:0, lauric C12:0, tridecanoicC13:0, myristic C14:0, pentadecanoic C15:0, palmitic C16:0,heptadecanoic C17:0, stearic C18:0, arachidic C20:0, heneicosanoicC21:0, behenic C22:0, lignoceric C24:0, myristoleic C14:1 n-5,cis-10-Pentadecenoic C15:1 n-5, palmitoleic C16:1 n-7,cis-10-heptadecenoic C17:1 n-7, elaidic C18:1 n-9 trans, oleic C18:1 n-9cis, cis-11-Eicosenoic C20:1 n-9, erucic C22:1 n-9, nervonic C24:1 n-9,linoelaidic C18:2 n-6 trans, linoleic C18:2 n-6 cis, gamma-linolenicC18:3 n-6, alpha-Linolenic C18:3 n-3, cis-11,14-Eicosadienoic C20:2 n-6,cis-8,11,14-eicosatrienoic C20:3 n-6, cis-11,14,17-eicosatrienoic 20:3n-3, arachidonic C20:4 n-6, cis-13,16-docosadienoic 22:2 n-6,cis-5,8,11,14,17-eicosapentanoic (EPA) C20:5 n-3,cis-4,7,10,13,16,19-Docosahexaenoic (DHA) C22:6 n-3 acid, lipoic acid,and combinations thereof.
 40. The method of claim 31, wherein themarkers further comprise an element marker of an element selected fromthe group consisting of lithium (Li), boron (B), magnesium (Mg),aluminium (Al), silicon (Si), phosphorous (P), sulfur (S), potassium(K), calcium (Ca), vanadium (V), chromium (Cr), manganese (Mn), iron(Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As),selenium (Se), bromine (Br), rubidium (Rb), strontium (Sr), molybdenum(Mo), tin (Sn), iodine (I), barium (Ba), titanium (Ti), sodium (Na),chlorine (Cl), and fluorine (F).
 41. The method of claim 31, wherein themarkers further comprise a vitamin marker of a vitamin or a metabolitethereof selected from the group consisting of vitamin B1, vitamin B2,vitamin B3, nicotinamide, methylnicotinamide, nicotinuric acid, choline,vitamin B5, vitamin B6, pyridoxal phosphate, pyridoxic acid, vitamin B8,vitamin B9, methyl tetrahydrofolic acid, vitamin B12, hydroxycobalamin,adenosylcobalamin, and combinations thereof.
 42. The method of claim 31,wherein the nucleotide marker comprises a marker of a nucleotideselected from the group consisting of inosine 5′monophosphate, adenosine5′monophosphate, cytidine 5′monophosphate, guanosine 5′-monophosphate,inosine 5′monophosphate, uridine 5′monophosphate, and combinationsthereof.
 43. The method of claim 31, wherein the osmolyte marker isselected from the group consisting of trimethylamine N-oxide,dimethylsulfoniopropionate, trimethylglycine, sarcosine, betaine,glycerophosphorylcholine, myo-inositol, and combinations thereof. 44.The method of claim 31, wherein the markers further comprise aphytonutrient marker of a phytonutrient selected from the groupconsisting of carotenoids, ellagic acid, flavonoids, chlorogenic acids,resveratrol, glucosinolates, phytoestrogens, or a combination thereof.45. The method of claim 31, wherein the peptide marker comprises amarker of a peptide selected from the group consisting of reducedgluthatione, oxidized glutathione, and combinations thereof.
 46. Themethod of claim 31, wherein the indirect marker is selected from thegroup consisting of combinations of nutrients and micronutrients,biomarkers of nutrients or micronutrients, metabolites of nutrients ormicronutrients, catabolites of nutrients or micronutrients,physiological states, ratios between nutrients and micronutrients,ratios between micronutrients, determinations of functional markers, andcombinations thereof.
 47. The method of claim 46, wherein the functionalmarkers comprise measurement of an activity selected from the groupconsisting of erythrocyte transketolase activity, erythrocyteglutathione reductase activity, oxidative stress status, nitric oxidesynthase activity, and combinations thereof.
 48. A method of treating ahuman subject suffering from a disease characterized by distinctivenutritional requirements in the human subject over a healthy subject,the method comprising: a. determining in a sample of the human subjectsuffering from the disease a profile of statuses of direct and indirectmarkers indicating the nutritional profile of the human subject, whereineach of the direct markers indicates an amount of a macronutrient ormicronutrient, and each of the indirect markers are derived from thedirect markers by quantifying a corresponding direct marker andquantifying one or more additional markers that indicate a status of thecorresponding direct marker, at least one of the direct and indirectmarkers is selected from the group consisting of an amino acid marker, afat marker, a carbohydrate marker, a nucleotide marker, an osmolytemarker, a peptide marker, a catabolism marker, a nitric synthase marker,and combinations thereof; b. determining in a sample of the healthysubject a profile of the statuses of the same markers comprising thesame direct and indirect markers determined in step a, at least one ofthe sample of the subject suffering from the disease or the sample ofthe healthy subject is a sample selected from the group consisting ofwhole blood, blood plasma, blood serum, red blood cells, urine, tissuebiopsies, and combinations thereof; c. comparing the profiles determinedin step a. and b., and determining distinctive nutritional requirementsfor nutrients comprising macronutrients and micronutrients in the humansubject suffering from the disease based on the comparison of theprofiles determined in steps a. and b., wherein when a level of themarkers determined in step a. is lower than a level of the same markersdetermined in step b., the distinctive nutritional requirements for thesubject comprise a composition comprising the nutrients in an effectiveamount to enable a level of the nutrients in the subject suffering fromthe disease to match a level of the nutrients in the healthy subject; d.providing the composition to restore the nutritional profile of thenutrients in the human subject suffering from the disease; and e.administering the composition to the human subject suffering from thedisease.